Fig 2: Increased recruitment of Mpx-positive neutrophils at the onset of regeneration after multiple cryoinjuries. (A) Cross sections of adult zebrafish hearts immunostained for L-plastin (green) and Myeloperoxydase (Mpx, red) at 4 dpci following one, two three and six cryoinjuries (CIs). The cardiac muscle is detected by F-actin staining (Phalloidin, blue). Both L-plastin and Mpx were detected in the wound. L-plastin/Mpx double positive cells are indicated (white arrow). The dashed line encircles the post-injury zone. Scale bars = 100 µm. (B) Analysis of the percentage of L-plastin-positive area within the wound. No significant change (NS) is observed between the experimental groups. N = 3. Scatter plot of the data with a large bar indicating the mean and smaller bars representing the SEM. (C) Analysis of the percentage of Mpx-positive area in the wound. Multiple cryoinjuries significantly increase the number of neutrophils compared to specimens after one cryoinjury. N = 3; *P < 0.05. Scatter plot of the data with a large bar indicating the mean and smaller bars representing the SEM.

Fig 3: M. circinelloides promotes neutrophils and macrophages depletion in the head kidney. (A) Representative sections of zebrafish head kidneys infected with 2.5 × 105 R7B spores/fish at 16 hpi and immunostained with anti-Lcp1 (pan-leukocyte marker) and anti-Mpx (neutrophil marker) antibodies. Note the robust depletion of myeloid cells. Scale bars 20 µm. (B) Quantification of Lcp1+ and Mpx+ cells is shown as the mean ± SEM of the immunostained area/total area of 4 randomly distributed optical areas from 4 fish at ×200 magnification. *P<0.05; **P<0.01; ***P<0.001.

Fig 4: M. circinelloides promotes the recruitment and apoptosis of macrophages in the infection site. (A) Representative serial sections of zebrafish head kidneys challenged with PBS, 2.5×105 R7B spores/fish and 107 (high dose, HD) NRRL spores/fish at 16 hpi and immunostained with anti-Lcp1, anti-Mpx, and anti-active Casp3 antibodies. Infiltrated leukocytes (black arrows) at 16 hpi were PAS+, Lcp1+ and most of them Casp3+ in the fish infected with the virulent strain (R7B) and the HD of the avirulent NRRL strain. Scale bars 20 µm. (B) Quantification of Lcp1+, Mpx+, and Casp3+ cells is shown as the mean ± SEM of the immunostained area/total area of 4 randomly distributed optical regions from 4 fish at ×200 magnification. *P<0.05.

Fig 5: Function of the cardiac lymphatic vasculature in regeneration.Whole-mount confocal imaging of adult transgenic zebrafish hearts expressing lyve1:RFP (White: A and B) and lyve1:GFP (Green: D-G) to mark lymphatic vessels. Additional fluorescence signal from injected Firefli Fluorescent Blue-dyed Microspheres (MA; blue: A and B), Quantum dots (Qdot; magenta: A and B) and immuno-labeled neutrophils (Mpx; white: D-G) and Macrophage (IB4; red:D-G). (A) Intra-myocardial injection of 200 nm microspheres (cyan) and 10 nm Qdots (magenta) results in both being deposited at the injection site (magenta/cyan arrowheads). Qdots, but not MS are also highly concentrated within the lymphatic vessels within the lyve1:RFP positive endothelium (arrowheads, A; uninjected control, B). (C) Schematic representation of nanoparticle uptake by the cardiac lymphatic system. (D, n = 5) Uninjured zebrafish heart labeled with antibodies against GFP (lyve1:GFP) and Mpx and lectinB4 (IB4), showing relatively few Mpx-positive neutrophils or IB4-positive macrophage on the ventricle or BA, including within the lymphatic vessels on the BA (green arrowheads). (E, n = 4) 1 day post-cryo-injured (dpc) heart (mild injury) labeled at the same time shows a massive increase in Mpx-expressing neutrophils and more moderate increases IB4-positive macrophage including within the lymphatic vasculature (green arrowhead (GFP) white arrowhead (Mpx) red arrowhead (IB4). Mpx-positive cells are concentrated along the visibly darker blood vessels (inset, blue arrowhead) away from the wound site (orange arrowhead). (F, n = 5) 7dpc heart showing persistent elevation of Mpx-positive and IB4-positive cells on the ventricle and BA, where they are associated with lymphatic vessels. (G) High resolution imaging of lyve1:GFP -positive vessels (green arrowhead, outlined in blue) on the BA of a 7dpc heart IB4-positive macrophage (red arrowhead) within the vessel and Mpx-positive neutrophil (white arrowhead) on the surface of the vessel (H) Graph showing quantification of increase in IB4-positive cells within the BA lymphatic vessels of 1dpc (n = 4) and 7dpc (n = 5) zebrafish hearts compared to those of uninjured hearts (n = 5). *p<0.05; **p<0.01, unpaired t-test, Mean and SD. Scale bars 200 µm (A-G) and 50 µm (right panels, D-F).Figure 5—source data 1.Source data for (H) and Supplement 1(G).